Production of loline alkaloids by the grass endophyte, Neotyphodium uncinatum, in defined media.

Lolines (saturated 1-aminopyrrolizidines with an oxygen bridge) are insecticidal alkaloids produced in symbioses of certain Epichloë (anamorph-Neotyphodium) species (fungal endophytes) with grasses, particularly of the genera Lolium and Festuca. Prior to the present study, it was unknown whether lolines were of plant or fungal origin. Neotyphodium uncinatum, the common endophyte of meadow fescue (Lolium pratense=Festuca pratensis) produced loline, N-acetylnorloline, and N-formylloline when grown in the defined minimal media at pH 5.0-7.5, with both organic and inorganic nitrogen sources and sugars as carbon sources. In contrast, lolines were not detected in complex medium cultures. GC-MS and 13C NMR spectroscopic analyses confirmed the identity of the alkaloids isolated from the defined medium cultures. Lolines accumulated to ca. 700 mg/l (4 mM) in cultures with 16.7 mM sucrose and 15-30 mM asparagine, ornithine or urea. Kinetics of loline production and fungal growth were assessed in defined medium with 16.7 mM sucrose and 30 mM ornithine. The alkaloid production rate peaked after the onset of stationary phase, as is common for secondary metabolism in other microbes.

Fungal endophyte-infected grasses: Alkaloid accumulation and aphid response.

The occurrence of the alkaloidsN-formyl andN-acetyl loline, peramine, lolitrem B, and ergovaline and the response of aphids to plants containing these compounds were determined in species and cultivars ofFestuca,Lolium, and other grass genera infected with fungal endophytes (Acremonium spp., andEpichloe typhina). Twenty-nine of 34 host-fungus associations produced one or more of the alkaloids, most frequently peramine or ergovaline. Three alkaloids (lolines, peramine, and ergovaline) were found in tall fescue and in perennial ryegrass infected withA. coenophialum, while peramine, lolitrem B, and ergovaline were present in perennial ryegrass and in tall fescue infected withA. lolii and inF. longifolia infected withE. typhina. WhileA. coenophialum andA. lolii produced similar patterns of alkaloids regardless of the species or cultivar of grass they infected, isolates ofE. typhina produced either no alkaloids or only one or two different alkaloids in the grasses tested. Aphid bioassays indicated thatRhopalosiphum padi andSchizaphis graminum did not survive on grasses containing loline alkaloids and thatS. graminum did not survive on peramine-containing grasses. Ergovaline-containing grasses did not affect either aphid.

Production of extracellular fibers by tobacco leaf epidermal protoplasts.

Protoplasts prepared from the upper epidermis of tobacco (Nicotiana tabacum L.) leaves produce tubular structures which are highly fluorescent in the presence of Calcofluor white. The presence of Ca(2+) in the incubation medium appears to be necessary for the production of these fibers. A commercial cellulase preparation destroyed these structures and inhibited their production.

Infection of tobacco leaf epidermal protoplasts with tobacco mosaic virus.

Protoplasts from the epidermal cells of tobacco leaves were prepared and inoculated with tobacco mosaic virus. Up to 50% of the protoplasts became infected and, within 3 days, contained an average of 5 x 10(5) virus particles per protoplast. Hexagonal crystalline inclusions were observed in many of the infected protoplasts.

Phloretinyl-3'-benzylazide: a high affinity probe for the sugar transporter in human erythrocytes. II. Irreversible transport inhibition is induced by photolysis.

In the dark, phloretinyl-3'-benzylazide (PBAz), at a nominal concentration of 10 microM, will inhibit the transport of D-glucose in human erythrocytes by more than 90%. This inhibition can be completely reversed by percolating the cell suspension through a small column of Sephadex G-10; cells recovered after this treatment, and then loaded with 100 mM D-glucose, possess a transport capacity (glucose efflux) equal to untreated cells. The Sephadex matrix completely removes non-covalently bound inhibitor even though, under these conditions (subdued light, 0.2% hematocrit, 0 degrees C, pH 6.2 or 7.8), from 70 to 80% of the PBAz added is bound to the cells (mostly non-specifically to hemoglobin). However, when erythrocytes exposed to 10 microM inhibitor are irradiated with long wavelength ultraviolet light, the glucose transporter is irreversibly inhibited; after 1 min irradiation, about 50% of transporter activity cannot be restored by Sephadex treatment. Under identical conditions, control cells (no PBAz, but irradiated and treated with Sephadex) retain over 90% of carrier activity. The photolytic conversion of the inhibition to an irreversible form is directly dependent on PBAz concentration. The results reaffirm our earlier conclusions that PBAz is a potentially useful photoaffinity labeling agent for the glucose transporter in erythrocyte membranes.

Phloretinyl-3'-benzylazide: a high affinity probe for the sugar transporter in human erythrocytes. I. Hexose transport inhibition and photolabeling of mutarotase.

A new phloretin derivative, phloretinyl-3'-benzylazide (PBAz), has been synthesized and compared with phloretin for its ability to inhibit the hexose transporter in human erythrocyte membranes in subdued light. Transport measurements were made using the light scattering (Orskov optical) method and a Millipore filtration technique with isotopically labeled sugars. Initial rates of sugar flux were measured under four different conditions to test for inhibition asymmetry. In each experimental condition, PBAz is from 6-20-times more potent than phloretin, making it one of the most effective reversible inhibitors known. Although both agents penetrate the cell membrane, they apparently fail to reach inhibitory levels at the inner surface over the time course of our nonequilibrated experiments, because of extensive binding to hemoglobin. The mechanism by which PBAz and its parent phloretin inhibit transport is pure competition with hexose for the carrier which faces the exterior of the membrane. If given time to equilibrate with the cells, the inhibition by both agents converts to a mixed type, i.e., both competitive and noncompetitive. The noncompetitive component could be due to inhibition of those transporter units oriented internally. Alternatively pre-equilibration with the inhibitors may cause them to attain high levels in the lipid membrane and produce nonspecific effects. PBAz and its precursor amine, phloretinyl-3'-benzylamine (PBA), compete with glucose for the sugar binding site on mutarotase at least as well as phloretin. When exposed to long wavelength ultraviolet radiation, PBAz is converted to a reactive intermediate which becomes covalently bound to the enzyme. Both irreversible ligand attachment and mutarotase inhibition are related to dose of the azide and irradiation time, but inactivation is from 5 to 6-times greater than label incorporation. We conclude that PBAz is a potentially useful photoaffinity labeling agent capable of covalently interacting with the transporter site facing the exterior of the red cell.

Kinetic advantage for transport into hamster intestine of glucose generated from phlorizin by brush border beta-glucosidase.

Phlorizin, labeled with tritium only in the glucose moiety, was used as substrate for the beta-glucosidase present in brush border membranes from hamster intestine in order to study, simultaneously, the kinetics of hydrolysis and the fate of the [3H]glucose liberated by the enzyme. The [3H]glucose seems to experience the same hydrolase related transport into the intestinal villi as the hexoses liberated from the common disaccharides byu their respective hydrolases. The released [3H]glucose accumulation rate is only partially inhibited by unlabelled glucose added to the medium as either the free sugar or as the precursors sucrose, lactose or glucose 1-phosphate, and then only when these sugars are present at very high levels. Furthermore, glucose oxidase, added to the medium as a glucose scavenger, has no effect on the uptake rate of the phlorizin hydrolase-liberated sugar. These and other findings are presented as evidence that, under conditions where the Na+-dependent glucose carrier is more than 97% inhibited by phlorizin, the glucose derived from the inhibitor, like the hexoses from disaccharides, has a kinetic advantage for transfer into the intestinal tissue.

A hydrolase-related transport system is not required to explain the intestinal uptke of glucose liberated from phlorizin.

The fate of [3H]glucose released from a wide range of [3H]phlorizin concentrations by phlorizin hydrolase has been studied under conditions where the Na+-dependent glucose transport system in hamster intestine is profoundly inhibited by the glucoside. At 0.2-2.0 mM phlorizin, the [3H]glucose uptake was a constant 11-12% of that generated by the enzyme and at the highest level, it was reduced to that of passive diffusion. Glucose liberated from 0.2 mM [3H]phlorizin is accumulated at a rate nearly equal to that found for 0.2 mM [14C]glucose when this free sugar uptake is measured in a medium containing 0.2 mM unlabeled phlorizin. Furthermore, without sodium, the accumulation rates of hydrolase-derived or exogenous glucose are both reduced to the rate of [14C]mannitol. Our results indicate that the glucose released from phlorizin enters the tissue via the small fraction of the Na+-dependent glucose carriers which escape phlorizin blockade together with a mannitol-like passive diffusion. It enjoys a kinetic advantage for tissue entry over free glucose in the medum by virtue of the position of the site where it is formed, i.e inside the unstirred water layer and near normal entry portals. No special hydrolase-related transport system, like the one proposed for disaccharides, needs to be considered to account for our findings.